Mesenchymal stem cell microsphere dressing derived from Fahrenheit gum and application of mesenchymal stem cell microsphere dressing

Abstract

The invention provides a Fahrenheit gum-derived mesenchymal stem cell microsphere dressing and application thereof.The Fahrenheit gum-derived mesenchymal stem cell microsphere dressing comprises Fahrenheit gum-derived mesenchymal stem cells, microspheres and a stem cell serum-free medium, the Fahrenheit gum-derived mesenchymal stem cells are from the second generation to the fifth generation of the mesenchymal stem cells, the content of the microspheres in the mesenchymal stem cell microsphere dressing is 30 wt%, and the content of the microspheres in the stem cell serum-free medium is 30 wt%. The content of the mesenchymal stem cells from the Fahrenheit gum in the mesenchymal stem cell microsphere dressing is 1 * 10 < 6 >-5 * 10 < 6 > / ml. The shape and preparation technology of gelatin are improved, the survival and proliferation of mesenchymal stem cells at wounds are promoted during use, the use of glutaraldehyde is avoided, and meanwhile, experimental results show that after the mesenchymal stem cell microsphere dressing derived from the Fahrenheit gum provided by the invention is dropwise added to the wounds, the wound healing rate is greater than 90% within 10 days, and the wound healing rate is greater than 90% within 10 days. Therefore, the mesenchymal stem cell microsphere dressing derived from the Fahrenheit gum, provided by the invention, has a relatively good repairing effect on the wound surface.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cells, and in particular to a mesenchymal stem cell microsphere dressing derived from Warren's jelly and an application thereof. Background Art

[0002] Mesenchymal stem cells exist in various tissue sites of adults, perinatal period or fetuses. They can be collected, isolated, expanded and cultured in vitro, and can differentiate into various types of tissue cells such as fat, bone, cartilage, muscle, nerve, skin, etc. Based on this, mesenchymal stem cells have been tried many times in the study of tissue and organ function damage and repair. Due to their potential impact on the wound microenvironment, they are considered to be the best candidate cells for cell regeneration therapy. Many studies have shown that mesenchymal stem cells promote wound healing by accelerating cell migration, wound closure, increasing angiogenesis, regulating inflammation at the wound site, promoting re-epithelialization and extracellular matrix remodeling. Although the therapeutic potential of mesenchymal stem cells in the process of skin wound healing has been confirmed, the delivery method of mesenchymal stem cells still needs further research and optimization. Intravenous injection or intradermal injection of mesenchymal stem cells to the wound site or around the wound is the main delivery method, but this method has low implantation efficiency and cells are retained in the wound site, which greatly reduces the repair effect of mesenchymal stem cells on the wound. The main It is composed of a mixture of polypeptide molecules with the same amino acid composition and a wide molecular weight distribution. It is formed by the partial degradation of collagen in connective tissues such as skin, bone, fascia, and myometrium of animal skin. It is white or light yellow, translucent, slightly shiny flakes or powder particles in appearance. It is a colorless, odorless, non-volatile, transparent, hard amorphous substance that is soluble in hot water. Gelatin also has good biocompatibility and can be completely absorbed and degraded by organisms without toxic side effects. Gelatin can promote cell adhesion and participate in cell migration, differentiation and proliferation. Therefore, it is widely used in medicine, beauty, trauma and other fields. Although the use of gelatin provides a good adhesion environment for cells, the block of gelatin is more viscous and long-term use may cause skin damage. In addition, gelatin is easy to degrade and is not easy to wrap cells to provide a stable extracellular matrix environment. In addition, the commonly used gelatin dressings inevitably use glutaraldehyde, formic acid and other cross-linking agents with certain toxicity. In this regard, this scheme specifically proposes a mesenchymal stem cell microsphere dressing derived from Wahler's jelly and its application to solve the above problems. Summary of the invention

[0003] In order to overcome the defects of the prior art, the present invention proposes a mesenchymal stem cell microsphere dressing derived from Fahrenheit jelly and its application to solve the problems raised in the above background technology.

[0004] To achieve the above object, the technical solution of the present invention is implemented as follows: a mesenchymal stem cell microsphere dressing derived from Warren's jelly and its application, wherein the mesenchymal stem cell microsphere dressing derived from Warren's jelly comprises mesenchymal stem cells derived from Warren's jelly, microspheres, a solvent and a stem cell serum-free culture medium.

[0005] Preferably, the Warburg jelly-derived mesenchymal stem cells are second to fifth generation mesenchymal stem cells, wherein when the composition is stored at about 25° C. for at least 6 months, at least about 80% of the mesenchymal stem cells contained therein remain viable.

[0006] Preferably, the content of the microspheres in the mesenchymal stem cell microsphere dressing is 30 wt %.

[0007] Preferably, the content of the mesenchymal stem cells derived from Wahler's jelly in the mesenchymal stem cell microsphere dressing is 2×10 5 ~2×10 6 The solvent is a suspension of the liquid medical biofunctional dressing, and the content of human albumin is 10-20 ng / ml.

[0008] The following steps are included:

[0009] S1. Prepare microspheres by weighing 1 g of gelatin with an electronic balance and adding it to 10 ml of deionized water. Stir magnetically at 30°C until the gelatin solution is completely dissolved to obtain a gelatin solution with a concentration of 10%;

[0010] S2. Add a certain amount of 100 mps dimethyl silicone oil as the oil phase into a beaker, then add 1 ml of span80 as an emulsifier, and preheat the mixture of dimethyl silicone oil and span80 to 60°C;

[0011] S3, slowly drop the gelatin solution preheated to 60°C into the oil phase, stir evenly, keep the speed constant, emulsify for a certain period of time in a 60°C water bath to form a uniform emulsion, after the emulsification is completed, cool the emulsion in an ice water bath, continue stirring during this process, keep the speed constant, add physiological saline for washing, and ultracentrifuge again for 0.5-2h, the centrifugal speed is 90000-100000 rpm;

[0012] S4, after cooling for a while, UV cross-linking and curing for 60 minutes to form a cross-linked polymer in the solution, stirring continuously during this process, the speed remains unchanged (the temperature is reduced to 5° C.), and the mesenchymal stem cell exosomes are added to the hyaluronic acid hydrogel step by step, so that the hyaluronic acid hydrogel encapsulates the mesenchymal stem cell exosomes;

[0013] S5. After the cross-linking is completed, pour 30 ml of supercooled acetone to perform dehydration and flocculation for 40 minutes to allow the microspheres to gradually solidify and settle. During this process, stirring is continued at a constant speed. Finally, the obtained mixed solution is washed 2 to 3 times with isopropanol to remove the oil phase on the surface of the microspheres, and then the diameter of the microspheres is measured;

[0014] S6. Extract mesenchymal stem cells from Warren's jelly. Healthy newborn umbilical cords are provided by the cooperating institution, Dongguan Binhaiwan Central Hospital. They are stored in a sterile and low-temperature environment and sent to the cell preparation laboratory within 3 hours. The Warren's jelly tissue is separated and cut into small pieces of 0.5mm2 for adherent culture. Ultra CULTURE medium is used for culture and subculture. The growth and morphology of the cells are observed in due course. After the growth and morphology are good and a certain number of third-generation (P3) mesenchymal stem cells with a viability of >90% are prepared, quality control is performed on them, including detection of bacteria, fungi, endotoxins, viability, surface markers, etc.

[0015] S7, preparation of dressings mixed with mesenchymal stem cells and microspheres derived from Warren's jelly, 2×10 5 ~2×10 6 Mesenchymal stem cells derived from Warren's jelly at a concentration of 100 wt % / ml were placed in a 15 ml sterile centrifuge tube, 30 wt % of the prepared gelatin microspheres were added, the microspheres and stem cells were gently mixed with a pipette to allow them to fully contact for suspension culture, the centrifuge tube cap was loosened and placed vertically in an incubator and cultured at 37° C. and 5% CO2 for 24 hours to obtain a mesenchymal stem cell microsphere dressing derived from Warren's jelly.

[0016] Preferably, a mesenchymal stem cell microsphere dressing derived from Warren's jelly is used in the skin wound healing process and provides a good adhesion environment for cells.

[0017] The beneficial effects of the present invention are embodied in:

[0018] The invention provides a mesenchymal stem cell microsphere dressing derived from Warren's jelly and application thereof. The invention innovatively adopts mesenchymal stem cells derived from Warren's jelly, improves the morphology and preparation technology of gelatin, promotes the survival and proliferation of mesenchymal stem cells at a wound when used, and avoids the use of glutaraldehyde. Meanwhile, experimental results show that after the mesenchymal stem cell microsphere dressing derived from Warren's jelly provided by the invention is dripped on a wound, the wound healing rate is greater than 90% in 10 days, indicating that the mesenchymal stem cell microsphere dressing derived from Warren's jelly provided by the invention has a good repairing effect on the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 It is a growth condition and morphology diagram of cells implemented according to the present invention;

[0021] Figure 2 It is a diagram of detecting cell surface markers according to the present invention;

[0022] Figure 3 is a functional diagram of three-lineage differentiation of cells implemented according to the present invention;

[0023] Figure 4 is a morphological diagram of microspheres prepared according to the present invention;

[0024] Figure 5 The diameter of the microspheres according to the present invention is 50-100 microns;

[0025] Figure 6 It is a healing rate diagram observed by microscopic imaging according to the present invention;

[0026] Figure 7 1 is a diagram showing the wound healing of mice according to the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments in the invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the invention.

[0028] It should be noted that if the embodiments of the invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0029] In addition, "multiple" means more than two. In addition, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, or it can be internal communication between two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the invention.

[0030] Example 1: Preparation of mesenchymal stem cells derived from Warren's jelly

[0031] The cooperative institution, Dongguan Binhaiwan Central Hospital, provided healthy newborn umbilical cords. The collection tube containing the umbilical cord was sprayed with an alcohol spray bottle and moved into a biosafety cabinet. The umbilical cord was then taken out with hemostatic forceps and placed in normal saline. The umbilical cord was rinsed and the blood clots attached to the surface of the umbilical cord, the blood clots in the blood vessels and the blood stains were removed as much as possible. A section of the head and tail was cut off with scissors. The cleaned umbilical cord was transferred to 70% medical alcohol for sterilization for a period of time, and then transferred to normal saline in time to rinse and remove 70% medical alcohol. The umbilical cord was cut into pieces in normal saline to obtain small pieces of umbilical cord tissue. The small pieces of umbilical cord tissue were inoculated into cell culture bottles, and the culture bottles were placed in a cell culture incubator for a period of time. After standing, mesenchymal stem cell culture fluid was added to the culture bottles, and the cells were cultured and subcultured with Ultra CULTURE medium, and the growth status and morphology of the cells were observed (see Figure 1 ), wait for the cells to grow to basic fusion, prepare a certain number of third-generation (P3) mesenchymal stem cells with high viability, and detect cell surface markers (see Figure 2 ) and cell lineage differentiation function (see Figure 3 ) and conduct quality control on it, including: bacteria, fungi, endotoxin, viability and other tests.

[0032] Example 2: Preparation of gelatin microspheres

[0033] (1) 1 g of gelatin was weighed using an electronic balance and added to 10 ml of deionized water. The mixture was stirred magnetically at 30° C. until the gelatin solution was completely dissolved to obtain a gelatin solution with a concentration of 10%.

[0034] (2) Add a certain amount of 100 mps dimethyl silicone oil as the oil phase into a beaker, then add 1 ml of emulsifier Span80, and preheat the mixture of dimethyl silicone oil and Span80 to 60°C.

[0035] (3) The gelatin solution preheated to 60°C is slowly added dropwise to the oil phase, stirred evenly, and the speed is kept constant. Emulsification is performed in a 60°C water bath for a certain period of time to form a uniform emulsion. After the emulsification is completed, the emulsion is cooled in an ice water bath. During this process, stirring is continued at a constant speed. After washing with physiological saline, ultracentrifugation is performed again for 0.5-2h at a centrifugal speed of 90,000-100,000 rpm.

[0036] (4) After cooling for a while, the solution was UV cross-linked and cured for 60 minutes to form a cross-linked polymer. During this process, stirring was continued at a constant speed (the temperature was reduced to 5° C.), and the mesenchymal stem cell exosomes were added step by step to the hyaluronic acid hydrogel so that the hyaluronic acid hydrogel encapsulated the mesenchymal stem cell exosomes.

[0037] (5) After the cross-linking is completed, 30 ml of supercooled acetone is poured into the solution for dehydration and flocculation for 40 minutes to allow the microspheres to gradually solidify and settle. During this process, stirring is continued at a constant speed. Finally, the obtained mixed solution is washed 2 to 3 times with isopropanol to remove the oil phase on the surface of the microspheres, and then the morphology of the prepared microspheres is observed (see Figure 4 ).

[0038] Example 3: Preparation of mesenchymal stem cell microsphere dressing derived from Wahler's jelly

[0039] (1) Take 2×10 5 ~2×10 6 Mesenchymal stem cells derived from Warren's jelly at a concentration of 100 μg / ml were placed in a 15 ml sterile centrifuge tube, 30 wt% of the prepared gelatin microspheres were added, and the microspheres and stem cells were gently mixed with a pipette to make them fully contact. The centrifuge tube cap was loosened and placed vertically in an incubator at 37°C and 5% CO2 for 24 hours. The diameter of the microspheres was 50-100 μm (see Figure 5 ).

[0040] Example 4: Application of mesenchymal stem cell microsphere dressing derived from Wahler's jelly

[0041] (I) Migration experiment

[0042] (1) Co-culture was performed using a polyester membrane Trans well with a pore size of 0.4 μm. The model cells were inoculated in the lower layer, and the upper chamber of the experimental group was inoculated with mesenchymal stem cell microsphere dressings derived from Warren's jelly; the negative control group was inoculated with only UltraCULTURE culture medium in the upper chamber.

[0043] (2) 6-well plate inoculated with 1.2×10 6 HDF cells.

[0044] (3) The next day, when the HDF cells adhered to the wall and the confluence reached 95%-100%, each well was scratched with a 10 μL pipette tip and the 6-well plate was gently rinsed with PBS three times.

[0045] (4) The experimental group was inoculated with 1×10 6 A small chamber of mesenchymal stem cell microsphere dressing derived from Warren's jelly was placed in a 6-well plate, and the dressing was co-cultured with the scratched HDF; the negative control group was co-cultured with the same volume of Ultra CUL TURE culture medium as the experimental group and the scratched HDF.

[0046] (5) Observe and calculate the healing rate through microscopic imaging at 0h, 12h, and 24h.

[0047] The experimental results are as follows Figure 6 As shown, the proliferation and migration speeds of cells co-cultured with the mesenchymal stem cell microsphere dressing derived from Fahrenheit Jelly are faster, which proves that the mesenchymal stem cell microsphere dressing derived from Fahrenheit Jelly of the present invention can promote the proliferation and migration of epidermal cells around the wound and promote the healing of the wound.

[0048] (II) In vivo experiments

[0049] (1) Twenty KM mice aged 5-8 weeks, half male and half female, were anesthetized with chloral hydrate and then depilated from the back;

[0050] (2) Use a hole puncher to make a 1-cm diameter circular hole on the left and right sides of the mouse's back, and fix the wound with a silicone gasket to prevent wound adhesion and healing;

[0051] (3) The left wound of the mice was the experimental group, and 0.1 mL of mesenchymal stem cell microsphere dressing derived from Warren's jelly was added. The right wound of the mice was the control group, and 0.1 mL of normal saline was added.

[0052] (4) Observe and record the wound healing status of mice every three days.

[0053] The experimental results are as follows Figure 7 As shown, compared with the wound surface dripped with normal saline, the wound surface dripped with the mesenchymal stem cell microsphere dressing derived from Fahrenheit Jelly increased the healing speed on the 6th day and was basically healed on the 9th day, which proves that the mesenchymal stem cell microsphere dressing derived from Fahrenheit Jelly of the present invention can promote the proliferation and migration of epidermal cells around the wound surface and promote the healing of the wound surface.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0055] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0056] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A mesenchymal stem cell microsphere dressing derived from Warren's jelly, characterized in that: The components of the mesenchymal stem cell microsphere dressing derived from Warren's jelly include mesenchymal stem cells derived from Warren's jelly, microspheres, a solvent and a stem cell serum-free culture medium.

2. The mesenchymal stem cell microsphere dressing derived from Warren's jelly according to claim 1, characterized in that: The Warburg jelly-derived mesenchymal stem cells are of the second to fifth passages, wherein when the composition is stored at about 25° C. for at least 6 months, at least about 80% of the mesenchymal stem cells contained therein remain viable.

3. The mesenchymal stem cell microsphere dressing derived from Warren's jelly according to claim 1, characterized in that: The content of the microspheres in the mesenchymal stem cell microsphere dressing is 30 wt %.

4. The mesenchymal stem cell microsphere dressing derived from Warren's jelly according to claim 1, characterized in that: The content of the mesenchymal stem cells derived from Fahrenheit jelly in the mesenchymal stem cell microsphere dressing is 2×10 5 ~2×10 6 The solvent is a suspension of the liquid medical biofunctional dressing, and the content of human albumin is 10-20 ng / ml.

5. A mesenchymal stem cell microsphere dressing derived from Warren's jelly according to any one of claims 1 to 4, characterized in that: The following steps are included: S1. Prepare microspheres by weighing 1g of gelatin with an electronic balance and adding it to 10ml of deionized water. Stir the gelatin solution by magnetic centrifugation at a temperature of 30°C until the gelatin solution is completely dissolved. The centrifugal speed is 90,000-100,000 rpm for 0.5-2h to obtain a gelatin solution with a concentration of 10%; S2. Add a certain amount of 100 mps dimethyl silicone oil as the oil phase into a beaker, then add 1 ml of span80 as an emulsifier, and preheat the mixture of dimethyl silicone oil and span80 to 60°C; S3, slowly drop the gelatin solution preheated to 60°C into the oil phase, stir evenly, keep the speed constant, emulsify for a certain period of time in a 60°C water bath to form a uniform emulsion, after the emulsification is completed, cool the emulsion in an ice water bath, continue stirring during this process, keep the speed constant, add physiological saline for washing, and ultracentrifuge again for 0.5-2h, the centrifugal speed is 90000-100000 rpm; S4. After spraying the surface of the fresh umbilical cord tissue with 75% ethanol, rinse it with a large amount of physiological saline immediately, cut off both ends of the umbilical cord with surgical scissors, drain the blood in the blood vessels with tweezers and clean it, cut the umbilical cord tissue into 3-5 cm long tissue segments with scissors (discard the edema part), divide the umbilical cord tissue into two parts along the artery with tweezers, remove two arteries and one vein, and peel off the Warthner's jelly with tweezers. Put the obtained Warthner's jelly into 6-10mL complete culture medium containing 10% platelet lysate and soak it at room temperature for about 30 minutes, scrape off the culture medium and mucus on the surface of Warthner's jelly with tweezers and place it in a 50mL centrifuge tube, cut it into 1-3mm tissue blocks with surgical scissors, evenly distribute the tissue blocks at the bottom of the T75 culture bottle, and culture it at 37℃. After 12 hours of culture, add 6 mL of complete culture medium containing 10% serum replacement to each T75 culture flask for culture. After that, replace the culture medium every 3 to 5 days. After the earliest three days of culture, cells can be observed to be free around the tissue blocks under the microscope. After 10 days of culture, the cell fusion degree can reach about 80%. Collect the tissue blocks and culture medium, centrifuge at 300g for 5 minutes, pour off the culture medium, and re-inoculate the tissue blocks into the T75 culture flask; S5. After the mixed emulsion of gelatin and naringenin is cooled for a while, the decellularized extracellular matrix particles are added and mixed, and the mixed solution is UV cross-linked and cured for 60 minutes to form a cross-linked polymer. During this process, stirring is continued at a constant speed (the temperature is reduced to 5° C.), and the mesenchymal stem cell exosomes are added step by step to the hyaluronic acid hydrogel, so that the hyaluronic acid hydrogel encapsulates the mesenchymal stem cell exosomes; S6. After the cross-linking is completed, 30 ml of supercooled acetone is poured in for dehydration and flocculation for 40 minutes to allow the microspheres to gradually solidify and settle. During this process, stirring is continued at a constant speed. Finally, the obtained mixed solution is washed 2 to 3 times with isopropanol to remove the oil phase on the surface of the microspheres, and then the diameter of the microspheres is measured; S7. Extract mesenchymal stem cells from Warren's jelly. Healthy newborn umbilical cords were provided by the cooperative institution, Dongguan Binhaiwan Central Hospital. The umbilical cords were stored in a low-temperature sterile manner and sent to the cell preparation laboratory within 3 hours. The Warren's jelly tissue was separated and cut into small pieces of 0.5 mm2 for adherent culture. Ultra CULTURE medium was used for culture and passage. The growth and morphology of the cells were observed in due course. After the growth and morphology were good and a certain number of third-generation (P3) mesenchymal stem cells with a viability of >90% were prepared, quality control was performed on them, including detection of bacteria, fungi, endotoxins, viability, surface markers, etc. S8, preparation of dressings mixed with mesenchymal stem cells and microspheres derived from Warren's jelly, 2×10 5 ~2×10 6 Mesenchymal stem cells derived from Warren's jelly at a concentration of 100 wt % / ml were placed in a 15 ml sterile centrifuge tube, 30 wt % of the prepared gelatin microspheres were added, the microspheres and stem cells were gently mixed with a pipette to allow them to fully contact for suspension culture, the centrifuge tube cap was loosened and placed vertically in an incubator and cultured at 37° C. and 5% CO2 for 24 hours to obtain a mesenchymal stem cell microsphere dressing derived from Warren's jelly.

6. A mesenchymal stem cell microsphere dressing derived from Warren's jelly as claimed in any one of claims 1 to 5, which is used in the process of skin wound healing and provides a good adhesion environment for cells.

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